In FY15, we have continued to analyze the tumor cell-targeted activities of TGF-beta, with a particular emphasis on effects on the cancer stem cell population. Our main experimental platform is a xenograft model of breast cancer progression based on the MCF10A human breast epithelial cell line. However, we have also established a number of new breast cancer cell strains from freshly excised patient tumors to extend our findings to primary cells. Our work in FY15 has focused in three main areas: 1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY15 we have continued deploy a novel functional imaging approach that we developed for the identification of the cancer stem cell (CSC) population. Our lentiviral-based CSC reporter uses a synthetic promoter in which expression of a fluorescent protein is driven by the stem cell master transcription factors Oct4 and Sox2. The reporter marks tumor cells that are enriched for CSC activities, including the ability to self-renew, divide asymmetrically, resist cell killing by conventional chemotherapeutics, and initiate tumorigenesis and metastasis in vivo. Using a xenograft breast cancer model in which TGF-beta functions as a tumor suppressor, we have combined our stem cell reporter with a TGF-beta pathway reporter and shown that endogenous TGF-beta signaling is activated more highly in the CSCs compared with the bulk population, suggesting a particularly important role for TGF-betas in the stem cell compartment. CSCs with endogenous TGF-beta signaling active were intrinsically less proliferative than CSCs that had not activated the pathway. Furthermore, we showed that TGF-beta selectively inhibited asymmetric self-renewing mitoses in the CSC compartment, and that it specifically inhibited the invasion of CSCs through basement membrane, while having little effect on the invasion of the bulk tumor cell population. These observations show that TGF-beta is an important modulator of CSC biology, and identify new mechanisms that could underlie the tumor suppressive effects of TGF-beta. We have optimized conditions for an ex vivo lung slice assay to analyze early stages of the metastatic process and probe the role of TGF-beta in early survival of disseminated cells and their metastatic outgrowth. We are also optimizing the system for intravital videomicroscopy to monitor stem cell properties in vivo in real time. Understanding how CSCs are regulated will be critical to development of more effective cancer therapies as these cells are largely resistant to existing therapeutic approaches. 2. MECHANISMS UNDERLYING THE CONTEXT-DEPENDENT EFFECTS OF TGF-beta ON THE CANCER STEM CELL COMPARTMENT. In our earlier work, we used ChIP-chip to identify genes in breast cancer that are regulated by TGF-beta and its downstream mediator Smad3. We showed that the constellation of TGF-beta/Smad3 target genes changed dramatically with tumor progression, suggesting that the readout of the TGF-beta signal is very sensitive to molecular context, as represented by the chromatin landscape and the spectrum of transcription factors and transcription co-regulators present in the cell at the specific stage in progression. Detailed analysis of Smad binding regions in the breast cancer models showed enrichment for the binding motif of a druggable transcription factor that may oppose the tumor suppressive effects of TGF-beta activity on the cancer stem cell. We are currently testing whether targeting this factor can restore TGF-beta-mediated tumor suppression in breast cancer models where this has been lost and we are analyzing underlying mechanisms. 3. DEVELOPMENT OF A DRUG SCREEN FOR CSCS. We are also using our cancer stem cell reporter to develop a medium-to-high throughput drug screen to test the NCI Natural Product Repository for novel agents that selectively inhibit the cancer stem cell.